P0131

Chondrocytes were seeded on glass coverslips overnight, fixed with 4% paraformaldehyde, premeabilized with 0.5% Triton‐X, and then blocked with 1% BSA. The cells were incubated with Col II (1:200, NB600‐844, Novus, MO), Aggrecan (1:100, NB600‐504, Novus, MO), and MMP13 (1:100, NBP1‐45723, Novus, MO) antibodies at 4°C overnight. Chondrocytes were then incubated with goat anti‐rabbit IgG Alexa Fluor 488 (A0423, Beyotime Biotechnology, Shanghai, China) or cy5 dye (P0183, Beyotime Biotechnology, Shanghai, China). Antifade mounting medium with DAPI (P0131, Beyotime, China) was used to stain nuclei.

Immunocytofluorescense staining was performed according to a previously described method.¹¹ Briefly, cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 at room temperature for 10 min. Next, the cells were incubated with 5% Bovine Serum Albumin (BSA) at room temperature for 1 h, followed by incubation with anti-CD90 (1:200; SC-53456, Santa Cruz), anti-TRPV4 (1:200; ab39260, Abcam), anti-GSDMD (1:200; AF4012, Affinity), anti-Caspase-1 (1:200; AF5418, Affinity), and anti-IL-1β (1:200; AF5103, Affinity) at 4 °C overnight. After thoroughly washed, the cells were then incubated with fluorescein isothiocyanate-conjugated or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (1:200; Zhongshan Golden Bridge Biotechnology, Beijing, China) in the dark at room temperature for 1 h. Nuclei were counterstained with DAPI (P0131, Beyotime, China). Confocal microscopic images were processed with LSM 5 Release 4.2 software after acquisition by a laser-scanning microscope (LSM510; Zeiss, Germany). The positively stained cells were counted in five different slides from each sample.

Treated cells were seeded on coverslips (Sarstedt Inc. TC coverslip 13 mm ST/CS200, Fisher Scientific) were fixed with 4% PFA in PBS for 15 min at room temperature. Cells were blocked with 1% BSA dissolved in PBS for 1 h after permeabilized with 0.1% Triton X-100 in PBS for 7 min, then incubated with primary antibody anti-5-mC (28692, Cell Signaling Technology) overnight at 4 °C, followed by Fluor 488-conjugated (1:200) incubation for 1 h at room temperature. The tablets were sealed with an anti-fluorescence quencher containing DAPI (P0131, Beyotime). IF images were acquired with a laser scanning confocal microscope (Andor) equipped with Imairs Viewer software.

The IF assays were tested as mentioned in our previous article [13 (link)]. Frozen sections of the patient's brain tissue were rewarmed. The cell membranes were permeabilized with 3% Triton X-100 followed by blocking with a blocking solution. The sections were incubated with the primary antibody overnight at 4°C, followed by the secondary antibody incubation at room temperature. Nuclei were stained with DAPI (P0131, Beyotime) containing an antiquencher, and then, the sections were photographed using a fluorescence microscope. The primary antibodies used were anti-Prx2 (10545-2-AP, Proteintech, 1 : 200), anti-NeuN (66836-1-Ig, Proteintech, 1 : 200), anti-Iba-1 (66827-1-Ig, Proteintech, 1 : 200), and anti-GFAP (60190-1-Ig, Proteintech, 1 : 200). The secondary antibodies used were CoraLite594-conjugated donkey anti-rabbit IgG (SA00013-8, Proteintech, 1 : 400) and CoraLite488-conjugated donkey anti-mouse IgG (SA00013-5, Proteintech, 1 : 400).

HK-2 cells were treated with rosiglitazone (100 μM) and TGF-β (20 ng/ml) for 48 h at 6-well dishes containing 25 mm coverslip per well. After treatment, the medium were removed and cells were washed three times with sterile PBS. Complete medium was used to prepare BODIPY 581/591 C11 (Thermo Fisher Scientific, D3681, USA) working solution with the concentration of 5 μM. 6-well dishes were added 1 ml per well and incubated at 37 °C for 30 min in the dark. At the end of the treatment, we removed the medium and cleaned cells three times with PBS. Glass coverslips with cells were fixed in 4% paraformaldehyde for 15 min. Subsequently, coverslips were transferred to glass microscope slide with the antifade mounting medium with DAPI (Beyotime, P0131, China). The images were captured using confocal microscopy (Nikon C2, Japan). To quantify the intensity of oxidization, the background was corrected by subtracting the red or green fluorescence in cell-free areas and then calculated the ratio of the green fluorescence to red and green fluorescence.

This experiment was grouped into control, US, HN-Ti₃C₂, and HN-Ti₃C₂+US groups. hBMSCs seeded on glass coverslips in 24-well plates were incubated for 14 or 21 days under the corresponding conditions according to the groups. The US and HN-Ti₃C₂+US groups were treated with US (1.0 MHz, 0.2 W/cm², 50% duty cycle) every two days for 10 min each time, for a total of 4 times. Cells were washed and fixed with 4% paraformaldehyde for 30 min before permeabilized with 0.5%Triton x-100 for 15 min. After sealing with blocking solution for 30 min, alkaline phosphatase (ALP; rabbit source, DF6225) and OPN (Affinity, rabbit source, AF0227) antibodies diluted in the antibody diluent were added to the corresponding wells and incubated for 12 h at 4 °C. After discarding the antibody, the wells were washed three times with 0.1% PBST and then anti-rabbit antibody (red fluorescence) (Proteintech, SA00013-4) was added to each well and incubated in the dark for 1 h at room temperature. Anti-rabbit antibody was discarded, and the wells were washed again with 0.1% PBST before staining the cytoskeleton and nucleus. The cytoskeleton was stained with green fluorescence-labeled phalloidin solution (Yeasen, 40735ES75), and the nuclei were stained with DAPI solution (Beyotime, P0131). Fluorescence staining images were captured using a fluorescence microscope.

The brains (n = 4/group) embedded with OCT were manufactured into 10-μm-thick frozen sections of TNC tissue in a cryostat microtome (Leica 1950 M). After fixation in ice-cold acetone, the sections were blocked by blocking buffer (10% normal goat serum, 0.5% Triton X-100, dissolved in 0.1 M PBS) for 1 h at room temperature (RT). Then, the sections were incubated with primary antibody (rabbit anti-GFAP antibody, 1:100, ab207165, Abcam) diluted in primary antibody dilution buffer (P0262, Beyotime) overnight at 4 °C. After cleaning with 0.1 M PBS three times, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, 1:1000, ab150077, Abcam) diluted in secondary antibody dilution buffer (P0265, Beyotime) for 1 h at RT. Then, the sections were rinsed in 0.1 M PBS three times and mounted with an antifade mounting medium with 4′,6-diamidino-2-phenylindole (DAPI, P0131, Beyotime). Magnified Images (× 20 objective) were captured under a fluorescence microscope (BX43, Olympus) using the cellSens standard software (version 1.18, Olympus). In negative-control sections, PBS was used instead of primary antibody, and there were no positive signals. The immunofluorescence area fraction was measured using ImageJ software (version 1.52p, National Institutes of Health).